Antibiotic resistance in bacterial pathogens is an immense and rapidly growing medical problem affecting the health of veterans and the broader population. Compounding this problem is the lack of new drugs, which together threaten our return to the pre-antibiotic era. The objective of this study is to molecularly and functionally characterize a novel and critical mediator of resistance to last-line polymyxin antibiotics, FlpR, which is conserved in numerous medically relevant bacterial pathogens. Importantly, FlpR also contributes to resistance to at least one host antimicrobial, as well as biofilm formation, and thus may play an important role in bacterial pathogenesis even beyond antibiotic resistance. Research Plan We will decipher how the FlpR protein contributes to antimicrobial resistance at the molecular level, by studying its effects on the structure of Gram-negative bacterial lipopolysaccharide (LPS), as well as elucidating its detailed mechanism of action. We will quantify the contribution of FlpR to resistance in the highly antibiotic-resistant nosocomial pathogen Acinetobacter baumannii, both in vitro and in in vivo mouse infections, also determining its contribution to virulence and biofilm formation. Methods: We will use a combination of genetic, molecular, and biochemical techniques to elucidate the mechanism of FlpR action as well as its contribution to A. baumannii physiology and resistance to antimicrobials. This will include a detailed study of its role in LPS biosynthesi, as well as the dissection of amino acids that are critical to its function. Clinical Relevance A. baumannii is a major nosocomial pathogen and cause of serious and life-threatening disease in veterans, soldiers, and civilians. Since FlpR is conserved in this and other clinically important pathogens (including Francisella spp., Bordetella spp., Brucella spp., Legionella pneumophila, Campylobacter spp., and Stenotrophomonas maltophilia), the insights gained in this proposal will have a broad impact on our understanding and treatment of bacterial disease. Furthermore, this work will lay the foundation for the potential future development of FlpR inhibitors, facilitating the treatment of even pan-resistant strains.